The present invention relates to a subfamily of RNA helicases, one of which is the MTT1 gene, which modulates the fidelity of translation termination. The present invention relates to a multiprotein surveillance complex comprising MTT1, human Upf1p, Upf2p, Upf3p, eucaryotic Release Factor 1 and eucaryotic Release Factor 3 which is involved in modulation of the efficiency of translation termination and degradation of aberrant mRNA. Identification of this complex provides an in vitro assay system for identifying agents that: affect the functional activity of mRNAs by altering frameshift frequency; permit monitoring of a termination event; promote degradation of aberrant transcripts; provide modulators (inhibitors/stimulators) of peptidyl transferase activity during initiation, elongation, termination and mRNA degradation of translation. Such agents which may be antagonists or agonists, are useful for screening, and diagnostic purposes, and as therapeutics for diseases or conditions which are a result of, or cause, premature translation.
The translational apparatus is responsible for synthesizing cellular proteins. This machinery must be able to determine the precise sites on the mRNA where decoding should begin and where it should end. The selection of the translation start site is usually delineated by the first AUG codon encoding the amino acid methionine. After initiation of translation, the ribosome manufactures the polypeptide by progressing along the mRNA in the 5xe2x80x2 to 3xe2x80x2 direction, decoding one codon at a time. The final step in the translation process occurs when one of three termination codons occupies the A-site of the ribosome, resulting in hydrolysis of the peptide (reviewed in Buckingham et al., 1997). Although translation termination normally occurs after completion of the full-length polypeptide, base substitutions and frameshift mutations in DNA often lead to the synthesis of an mRNA that contains an inappropriate stop codon within its protein coding region. The occurrence of such a premature stop codon arrests translation at the site of early termination and causes the synthesis of a truncated protein and rapid degradation of the mRNA (reviewed in Ruiz-Echevarria et al., 1996; Weng et al., 1997). Interestingly, nonsense and frameshift mutations cause approximately 20-40% of the individual cases of over 240 different inherited diseases (reviewed in McKusick, 1994). Thus, treatment of a number of genetic disorders can be envisioned by promoting nonsense suppression. Nonsense suppression results when a near cognate tRNA successfully competes with the termination factors at a nonsense mutation so that amino acid incorporation into the peptide chain occurs rather than prematurely terminating translation (FIG. 1). Sufficient levels of nonsense suppression allows production of completed polypeptide protein. For many diseases in which only one percent of the functional protein is produced, patients suffer serious disease symptoms, whereas boosting expression to only five percent of normal levels can greatly reduce the severity or eliminate the disease (McKusick, 1994; Cooper etc.). Recent reports have demonstrated that sub-inhibitory concentrations of certain aminoglycosides suppress the translation termination process, resulting in the expression of full-length CFTR and restoring cyclic AMP-activated chloride channel activity (Bedwell et al. 1997; Howard et al., 1996). Thus, identifying and characterizing the factors that regulate the efficiency of the translation termination will be important for understanding the biology of this process as well as in developing therapeutics for the treatment of a wide array of genetic disorders that arise as a consequence of a nonsense mutations.
Translation termination is carried out by the eucaryotic peptidyl release factors Release Factor 1 (eRF1) and Release Factor 3 (eRF3). Both eRF1 and eRF3 are conserved proteins that interact and promote peptidyl release in eucaryotic cells (Frolova et al. 1994, Stansfield et al. 1995, Zhouravleva et al. 1995). In yeast, eRF1 and eRF3 are encoded by the SUP45 and SUP35 genes, respectively (Frolova et al. 1994, Zhouravleva et al. 1995). Sup45p (eRF1) and Sup35p (eRF3) have been shown to interact (Stansfield et al. 1995, Paushkin et al 1997a,b). eRF1 contains intrinsic peptide hydrolysis activity while eRF3, which has homology to the translation elongation factor EF1xcex1 (Didichenko et al. 1991), demonstrates GTPase activity (Frolova et al. 1996), and enhances the termination activity of eRF1 in a GTP-dependent manner (Zhouravleva et al. 1995).
Factors that modulate the efficiency of translation termination process have been identified (Weng et al., 1996a,b; Czaplinski et al., 1998; Song and Liebman, 1987; All-Robyn et al. 1990). For example, recent results indicate that the Upf1p is a factor that modulates the efficiency of translation termination. Disruption of the UPF1 gene results in a dramatic stabilization of nonsense-containing mRNAs and promotes suppression of certain nonsense alleles (Leeds et al. 1991, Cui et al. 1995, Czaplinski et al. 1995,1998 Weng et al. 1996a,1996b). Recent results suggest that the Upf1p may modulate the translation termination process by directly interacting with eRF1 and eRF3 (Czaplinski et al., 1998). The Upf1p contains a cysteine- and histidine-rich region near its amino terminus and all the motifs required to be a member of the superfamily group I helicases (Czaplinski et al. 1995,; Weng et al. 1996a,b, 1998, Altamura et al. 1992, Cui et al. 1996, Koonin, 1992, Leeds et al. 1992, Atkin et al. 1995,1997). The yeast Upf1p has been purified and demonstrates RNA-dependent ATPase and helicase activity (Czaplinski et al. 1995, Weng et al. 1996a,b, 1998). A human homologue of the UPF1 gene, called RENT1 or HUPF1 (Perlick et al. 1996, Applequist et al. 1997) has been identified and shown to be functional in yeast cells in enhancing translation termination, indicating that its role in this process is evolutionarily conserved (Czaplinski et al., 1998).
The results presented here identify a set of superfamily group I helicases in yeast cells with significant homology to Upf1p. In particular, one gene and its protein product called MTT1 (for Modulator of Translation Termination) has been characterized. Mtt1p encodes a superfamily group I helicase and harbors a cysteine-histidine-rich region in its amino terminus. Similar to Upf1p, Mtt1p interacts with the translation termination factor eRF3 and can modulate the translation termination process. Significantly, inactivation of both Upf1p and Mtt1p demonstrate a dramatic nonsense suppression phenotype that is greater than the nonsense suppression phenotype observed for either deletion. These results demonstrate that there is a family of RNA helicases that are modulators of the translation termination process.
This invention provides a method for identifying a test composition or agent which modulates the efficiency of translation termination which comprises: (a) contacting MTT1 with a test composition under conditions permitting binding between MTT1 and the test composition; (b) detecting specific binding of the test composition to the MTT1; and (c) determining whether the test composition inhibits the MTT1 so as to identify a test composition which is which modulates the efficiency of translation termination. In one embodiment, the agent inhibits ATPase/helicase activity of MTT1, ATPase of Upf1p; GTPase activity of eRF1 or eRF3; RNA binding; binding of the factors to the ribosome; or binding of the factors to each other. In another embodiment the agent modulates the binding of MTT1 to the polysome. In another embodiment the agent inhibits the binding of human MTT1 to eRF3. In another embodiment the agent facilitates the binding of human MTT1 to eRF3.
This invention provides a method of identifying a test composition or agent which modulates binding to MTT1, the method comprising: (a) incubating components comprising the test composition, and MTT1 wherein the incubating is carried out under conditions sufficient to permit the components to interact; and (b) measuring the effect of the test composition on the binding to MTT1. In one embodiment the method further comprising identifying a gene comprising; (a) introducing into a cell a test composition which modulates binding to MTT1; (b) determining the phenotype of the cell after (a); (c) comparing the cellular phenotype after (a) with the cellular phenotype before (a); and (d) identifying the gene of the cell into which the test composition has been introduced.
This invention provides a vector which modulates the expression of MTT1 polynucleotide or the function of MTT1 polypeptide. In one embodiment the modulation is inhibitory. In another embodiment the modulation is stimulatory.
This invention provides an isolated multiprotein complex comprising a MTT1 gene, human Upf1p protein, a peptidyl eucaryotic release factor 1 (eRF1) and a peptidyl eucaryotic release factor 3 (eRF3), wherein the complex is effective to modulate peptidyl transferase activity during translation. In one embodiment the complex further comprising human Upf3p and/or Upf2p.
This invention provides an agent which binds to the complex which modulates the fidelity of translation of an mRNA. Translation includes initiation, elongation, termination as well as degradation. In one embodiment, the agent inhibits ATPase/helicase activity of MTT1, ATPase of Upf1p; GTPase activity of eRF1 or eRF3; RNA binding; binding of the factors to the ribosome; or binding of the factors to each other. In another embodiment the agent modulates the binding of MTT1 to the polysome. In another embodiment the agent inhibits the binding of human MTT1 to eRF3. In another embodiment the agent facilitates the binding of human MTT1 to eRF3.
This invention provides a method of modulating peptidyl transferase activity during translation, comprising contacting a cell with the agent, in an amount effective to suppress nonsense translation termination, thereby modulating the peptidyl transferase activity. The peptidyl transferase activity during translation occurs during initiation, elongation, termination and degradation of mRNA.
This invention provides a method of modulating the efficiency of translation termination of mRNA at a nonsense codon and/or promoting degradation of aberrant transcripts, comprising contacting a cell with the agent, in an amount effective to inhibit the binding of Mtt1 and eRF3, thereby modulating the efficiency of translation termination of mRNA at a nonsense codon and/or promoting degradation of aberrant transcripts.
This invention provides a method of modulating the efficiency of translation termination of mRNA at a nonsense codon and/or promoting degradation of aberrant transcripts, comprising contacting a cell with an agent, which inhibits the ATPase/helicase activity of MTT1, thereby modulating the efficiency of translation termination of mRNA at a nonsense codon and/or promoting degradation of aberrant transcripts.
This invention provides a method of detecting a disorder associated with the expression of Mtt1 protein, wherein the method comprises contacting a sample from a subject having or suspected of having a disorder with a reagent that detects expression of the mtt1 protein or mutant thereof and detecting the binding of the reagent in the sample.
This invention provides a method for treating a disease associated with peptidyl transferase activity, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the complex, mtt1 protein, mutant mtt1 protein, or agents thereto, and a pharmaceutical carrier or diluent, thereby treating the subject.
This invention provides a method of identifying genes which are involved in modulation of the fidelity of translation termination, which comprises: a) isolated a gene of interest; and b) determining whether the gene of interest comprises motifs I-IX, wherein if the gene comprises any one of the nine motifs the gene modulates translation termination. In one embodiment motif I comprises the sequence: GppGTKTxT-X(n) (SEQ ID NO:1). In another embodiment motif II comprises the sequence riLxcaSNxAvDxl-X(n) (SEQ ID NO:2). In another embodiment motif III comprises the sequence vviDExxQaxxxxxiPi-X(n) (SEQ ID NO:3). In another embodiment motif IV comprises the sequence xxi1 aGDxxQLp-X(n) (SEQ ID NO:4). In another embodiment motif V comprises the sequence lxx SLF erv-X(n) (SEQ ID NO:5). In another embodiment motif VI comprises the sequence LxxQYRMhpxisefpxYxgxL-X(n) (SEQ ID NO:6). In another embodiment motif VII comprises the sequence IgvitPYxxQvxxl-X(n) (SEQ ID NO:7). In another embodiment motif VIII comprises the sequence vevxtVDxFQGreKdxlilSc VR-X(n) (SEQ ID NO:8). In another embodiment motif IX comprises the sequence iGFLxdxRRINVaITRak.